To search for novel paracrine modulators of heart cell function. We studied the effects of effluent of superfused of normoxic and hypoxic endocardial and vascular endothelial cultured cells on contraction and intracellular calcium transients of isolated adult rat cardiac myocytes. We found that EEC synthesize and release endothelin, which accounts for a positive inotropic effect of sheep EEC effluent. Additionally, both endocardial and vascular endothelial cells tonically release a novel substance which rapidl and reversibly decreases the amplitude of myocytes twitch contraction by inducing earlier relaxation, and also increase diastolic cell length. Thes effects are not associated with any change in the intracellular calcium transient, indicating cardiac myofilament "desensitization". The activity of endothelial cell effluent remained stable at 37xC for several hours or a 4xC for at least 48 hours. The action of this substance did not involve nitric oxide, cyclic GMP or prostanoids, nor changes in intracellular pH. These properties suggest that endothelial cells may rapidly modulate cardia contraction-relaxation coupling and diastolic tonus by altering myofilament properties, as well as exert distant effects because of the unusual stability of this substance. Superfusates of hypoxic endocardial and vascular endothelial cells induced rapid, reversible reduction in myocyte twitch amplitude (-67.0 q 4.9%; mean q S.E.M.), and decreased diastolic length (-1.5q0.3fm; both P <0.001; n=18). Ca2+ transients were however unchanged indicating altered myofilament properties. This effect was not attributable to known endothelial agents, nor to changes in pH1 in SNARF- loaded myocytes. Superfusate of hypoxic cells (to 1/20 dilution), but not normoxic cells, completely and reversibly inhibited the in vitro sliding of F-actin over rat cardiac myosin, and dramatically reduced actin-activated myosin S1 ATPase activity. Neither effect was observed with smooth muscle myosin. These data suggest a novel endothelial cell-mediated feed forward mechanism which senses hypoxia and depresses myocyte contraction, by releasing factors(s) that directly inhibit crossbridge cycling. This may explain the clinical phenomenon of myocardial hibernation, i.e., depressed contraction of viable myocardium during reduced coronary flow.